1. Field of the Invention
The present invention relates to a vector system useful for developing live viral vaccines. More particularly, it relates to replication-competent recombinant Sabin type 1 strain of poliovirus, which can be used for the development of live viral vaccine capable of inducing mucosal immunity.
2. Description of the Prior Arts
Recently it has been reported that various infectious viral disease, which were well known to be spread by blood-mediated routes such as blood transfusions, homosexual intercourse, or sharing of, syringes, also may be transmitted by heterosexual intercourse. In the case of AIDS (Acquired Immunodeficiency Syndrome), the number of heterosexual transmission is far greater than that of the blood-mediated cases (Stingl et al., J. Am. Aca. Dermatol., 22, 1210, 1988). In Korea, 363 out of 527 HIV-1 positive patients are heterosexuals while 99 are infected via homosexual routes (National Institute of Health, Korea, Communicable Diseases Monthly Report, April of 1996). These reports strongly suggest that the HIV-1 can be transmitted through mucosal tissues around the genital organs without blood mediation.
Several papers have reported that HIV-1 transmission and spreading are likely to be initiated by the infection of Langerhans cells or dendritic cells (DCs) at the mucosal tissues. Infected cells return to lymph node to deliver antigen, well known as homing property, where the viral replication occurs, resulting in viremia and AIDS progression. In other words, those who have heterosexual intercourse with HIV-1 infected patients will have Langerhans cells or DCs infected with HIV-1 in mucosal area of the urogenital organs, and the infected DCs return to lymph nodes, activate CD4+ T-cells in lymph nodes and propagate HIV, resulting in depletion of CD4+ T-cells followed by AIDS progression (Tschachler et al., J. Inves., Dermator, 88, 238, 1987; Langhoff et al., Proc. Natl.Acad.Aci. USA, 88, 7998, 1991; Patterson and Knight, J. Gen. Virol., 68, 1177, 1987; Patterson et al., Immnol., 72, 361, 1991; Cameron et al., Science, 257, 383, 1992; Embreston et al., Nature, 362, 369, 1993; Fauci, Science, 262, 1011, 1993; Pantleo et al., Nature, 362, 355, 1993; Adema et al., Nature, 387, 713, 1997). These experimental results were substantiated by in vivo experiments by addressing that the Macaque monkeys treated with SIV (Simian immunodeficiency Virus) on their genital organs became infected and then showed AIDS symptom (Miller and Gardner, J. AIDS, 4, 1169, 1991; Miller et al., 3. Virol, 63, 4277, 1989). Moreover, since most of the infectious viral diseases are spread by first infection of mucosal tissues at respiratory, digestive or urogenital organs, mucosal vaccine development is highly recommended to prevent infectious viral. In particular, the common mucosal immune systemxe2x80x94immunization at one locus can induce identical immunity to the other mucosal areas in the living body, special characteristics of mucosal immunity, encourages many researchers to develop mucosal vaccine (Kott, Science, 266, 1335, 1994; Cease and Verzofsky, Ann. Rev. Immunol., 12, 923, 1994).
Since long ago, smallpox virus has been proposed as a live viral vaccine vehicle. Recombinant vaccinia virus produced by introducing a vaccine gene into smallpox viral genome was reported to induce cytotoxic T lymphocyte in the immunized monkey. But has not yet been allowed to apply it to human because it may cause a vaccinia syndrome to the immunized individuals when overpropagated. To avoid the possibility, an attenuated vaccinia virus was suggested as a vaccine vehicle instead of virulent strain, but it failed to induce an effective immuniity (Cooney et al., Lancet, 337, 567, 1991; Tartaglia et al., Virol., 188, 217, 1992).
Adenovirus having a smaller genome (34 kbp) than that of vaccinia virus was also proposed as a vaccine vector (Natuk et al., Proc. Natl. Acad. Sci. USA, 89, 7777, 1992; Gallichar et al., J. Infec. Dis., 168, 622, 1993). But the recombinant adenoviruses still have a limitation of side effects, such as conjuctivitis or corneitis, which should be solved for adenovirus to be used as a mucosal vaccine vector.
Poliovirus contains a positive sense single-stranded RNA of 7.4 Kb nucleotides, which encodes an unique open-reading frame of a long polyprotein (Kitamura et al., Nature 291, 547, 1981). Recently, several groups are trying to develop a poliovirus as a vaccine vehicle for its well-known and attractive advantagesxe2x80x94safe, easy to administration, economy, and above all having capacity to induce effective life-long mucosal immunity, which is strongly recommended for an ideal vaccine.
Followings are summary of the published vaccine researches in association with poliovirus developed as a vaccine vehicle:
(1) It was proposed to substitute some portion of VP1, major outer capsid protein of poliovirus with presumed vaccine epitopes of HIV such as gp41, PND (Principle Neutralizing Domain) or gp120. The chimeric virus produced from the genetic recombination effectively induced antibody depending on the characteristics of the epitopes (Burke et al., Nature, 332, 81, 1988; Burke et al., J.Gen.Virol. 70,2475, 1989; Evans et al., Nature, 385, 1989; Dedieu et al., J. Virol., 66, 3161, 1992; Rose et al., J. Gen. Virol., 75, 969, 1994). However, this chimeric virus has a size limitation for the Morrow and his introduced vaccine gene. Chimeric poliovirus could not be assembled properly in the infected cells when the inserted vaccine epitope is larger than 25 amino acid residues.
(2) Morrow and his colleagues (Porter et al., J. Virol., 69, 1548, 1993; Ansaradi et al., Cancer Res., 54, 6359, 1994, Porter et al., J. Virol. 70, 2643, 1996, Porter et al., Vaccine 15, 257, 1997) have suggested poliovirus minireplicon, in which poliovirus structural genes are replaced by foreign sequences, to develop poliovirus-mediated mucosal vaccines. In case of poliovirus minireplicon, the replication defective recombinant viral genome must be co-transfected with other capsid protein-expressing vector for packaging of chimeric viral genome (Porter et al., J. Virol., 69, 1548, 1995). Moreover, high titer of minireplicon is required for vaccination to induce effective mucosal immunity because it works as replication-defective target-specific immunogen rather than live viral vaccine.
(3) Recently, a new strategy was suggested for expression of foreign antigens in the replication-competent recombinant polioviruses by Mattion et al (J. Virol. 68, 3925, 1994) and Andino et al (Science, 265, 1448, 1994). They have introduced a new polylinker region and 3C protease-recognition site on the N-terminal end of the polyprotein of poliovirus. According to this system, foreign gene, cloned in-frame with the poliovirus open reading frame, is followed by an artificial 3C protease site, to allow proteolytic cleavage of the foreign protein from the poliovirus polyprotein. The exogenous nucleic acid is incorporated directly into the poliovirus genome. The exogenous sequences are expressed during virus replication as part of the virus polyprotein and subsequently processed by virus-encoded proteases to produce free antigen and mature viral protein. The foreign antigen is not packaged in the virion but released into the cytoplasm. The prinicle of this method is based on the characteristics of poliovirus-specific 3C-protease published previously. 3C-protease recognizes specific amino acid sequence and then cleaves it at the junction between Glu(Q)/Gly(G) (Hanecak et al., Proc. Natl. Acad. Sci. USA 79, 3793, 1982), and the proteolysis occurs within the intramolecule of long polyprotein (Palmenberg and Reuckert, J. Virol., 41, 244, 1982; Hanecak et al, Cell 37, 1063, 1984). These phenomena are generally observed in picornavirus family (Palmenberg et al., J. Virol. 32, 770, 1979; Palmenberg and Reuckert, J. Virol., 41, 244, 1982). The alanine(A) residue in the P4 position of the Q/G cleavage site (AXXQG; SEQ ID NO: 21) has been confirmed several times to be essential for effective recognition and cleavage by 3C-protease (Nicklinson et al., Biotechnology 4, 33, 1986; Pallai et al., J. Bio. Chem., 264, 9738, 1989; Cordingley et al., J. Virol., 63, 5037, 1989; Orr et al., J. Gen. Virol., 70, 2931, 1989; Petithory et al., Proc. Natl. Aca. Sci. USA 88, 11510, 1991; Blair and Semler, J. Virol. 65, 6111, 1991). Based on these experimental results, Mattion and his colleagues introduced (J. Virol. 68, 3925, 1994) multiple cloning site and 3C-protease cleavage site into N-terminal of Sabin type 3 strain of poliovirus and constructed chimeric poliovirus expressing rotavirus VP7. Andino and his colleagues (Science, 265, 1448, 1994) constructed recombinant Mahoney vector (MoV-1.4) by introducing multiple cloning site and 3C-protease cleavage site into the N-terminal end of the long polyprotein of poliovirus Mahoney strain. They produced chimeric poliovirus by cloning various HIV-1 subgenomes into the vector (Science, 265, 1448, 1994). Andino group has reported that the chimeric poliovirus expressing HIV-1 nef gene induced effective mucosal immunity in monkey two weeks after immunization through rectum (Andino et al., Science, 265, 1448, 1994). Nevertheless, the recombinant Mahoney poliovirus is not applicable to human without further detoxification steps since Mahoney strain of poliovirus is a deadly virulent neurotropic virus, which infects central nervous system of primates through the primary infection of digestive organ, resulting in causing paralytic poliomyelitis. Whereas, Meuller and Wimmer (J. Virol., 72, 20-31, 1998) reported that the recombinant virus obtained by cloning green fluorescence protein gene (gfp:252aa) and HIV-1 gag gene into Andino""s Mahoney vector were not so much genetically stable during the passages as reported previously by Andino et al. (Science, 265, 1448, 1994). Their experimental results revealed that most of the recombinant viruses even after a single passage lost the introduced vaccine gene, and none of the progeny virus after 6 passages has been found to have full-length exogeneous insert in RT-PCR experiment.
(4) In 1996, Andino and his colleagues also reported recombinant Mahoney virus expressing preS (118aa, 54aa) and core (155aa) proteins of hepatitis B virus using the same Mahoney vector (Yim et al., Virology, 218, 61, 1996). Even though they have stated that these chimeric viruses were genetically stable during the passages until the sixth progeny, their RT-PCR experimental result showed that the amounts of recombinant virus maintaining preS gene (118 aa) were markedly reduced during the fifth passage. On the other hand, their report revealed that the recombinant virus expressing a small preS protein coding 55aa residues had a markedly reduced replication capacity as compared with that of wild-type or other recombinant Mahoney strains. The result suggests that the replication capacity of recombinant chimeric viruses is not so much tightly associated with the size of the inserted vaccine gene as expected. These experimental results lead us to conclude that the biological characteristics of the recombinant chimeric virus depends on several combined factors like the size of gene inserts, characteristics of the vial vector, introduced vaccine genes, etc.
(5) Andino et al., accepting the problems of their Mahoney vector as mentioned above, developed a new Mahoney vector (MoV-2.1) by inserting a multiple cloning site and 2A-protease cleavage site at P1/P2 junction of Mahoney polyprotein cDNA (Tang et al., J. Virol., 71, 7841-7850, 1997). This MoV-2.1 vector was employed to construct recombinant chimeric virus expressing SIV gag genes (p17 or p27) or env genes (gp130 or gp41). These recombinant chimeric viruses have the similar replication capacity to that of wild type Mahoney, and express the introduced vaccine gene efficiently. However, the MoV-2.1 vector still has a problem of genetic instability. Over 99% of the recombinant chimeric viruses lose the introduced vaccine genes within the third passage. They thought that the sequence deletion during the passages was to be due to the homologous recombination between the repeated sequences at the newly introduced multiple cloning sites. They have reduced the sequence homology by 37% at the repeated sequence of Mov-2.1 vector by performing silent mutation to change the sequence without affecting amino acid sequence, and then named it MoV-2.11. They have constructed the recombinant chimeric virus Sp27(MoV-2.11) by cloning SIV p27 gene into the manipulated Mahoney vector (MoV-2.11). For the recombinant virus without silent mutation [Sp27(MoV-2.1)], 20-30% of the plaques maintained the cloned vaccine gene after first passage and none of the plaques after third passage carried the cloned gene. On the other hand, for the recombinant virus with silent mutation at the repeated sequence [Sp27(Mo-2.11)], more than 90% of the single-passaged plaques maintained the cloned gene (gag) and 30-50% of the plaques expressed the SIV gene product at third passage. Nevertheless, the Mahoney vector MoV-2.11 still has a problem of genetic instability during the passages, even though they have some progress to increase genetic stability of Mahoney vector by reducing sequence homology. According to their experimental results, the population of insert-maintaining progeny viruses was markedly reduced as passage goes on even within three passages; first (90%), second (50-70%), and third (30-50%). That means that the insert-maintaining recombinant, chimeric Mahoney virus will be rapidly diluted out among the total population if passaged little bit further.
Considering several limitations of Mahoney vectors developed by Andino group, the Mahoney vector can not be allowed as a model system for the poliovirus-mediated mucosal vaccine vector.
Therefore, it is highly recommended to develop a new model system for poliovirus-mediated mucosal vaccine vectors, which is able to overcome the limitations shown in Mahoney vectors. For that purpose, the new vector should meet the following requirements: (1) viral vector should be safe to human beings; (2) recombinant virus should be replicable, and have equivalent replication capacity to that of wild type; and (3) the introduced vaccine genes should be stably maintained during the viral passages.
An object of the present invention is to provide a replication-competent recombinant Sabin 1 poliovirus vector that is useful as a live viral vaccine vector for the development of mucosal vaccines.
Another object of the present invention is to provide a chimeric poliovirus expressing several vaccine genes cloned into the multiple cloning site of the recombinant Sabin 1 poliovirus.
The present invention provides a replication-competent recombinant Sabin 1 poliovirus vector having nongenomic sequences coding for multiple cloning site and 3C-protease cleavage site between the first amino acid and second amino acid of long polyprotein of Sabin 1 poliovirus cDNA.
The present invention further provides a replication-competent chimeric Sabin 1 poliovirus expressing exogenous vaccine genes, respectively, and their recombinant cDNA plasmids, where which have exogeneous vaccine genes, respectively, at the multiple cloning site of the above-mentioned replication-competent recombinant Sabin 1 poliovirus vector.
The objects mentioned above, other features and applications of the present invention would be much more apparent by those of ordinary skills in the art from the following explanation in detail.